The pCri System: A Vector Collection for Recombinant Protein Expression and Purification

Abstract

A major bottleneck in structural, biochemical and biophysical studies of proteins is the need for large amounts of pure homogenous material, which is generally obtained by recombinant overexpression. Here we introduce a vector collection, the pCri System, for cytoplasmic and periplasmic/extracellular expression of heterologous proteins that allows the simultaneous assessment of prokaryotic and eukaryotic host cells (Escherichia coli, Bacillus subtilis, and Pichia pastoris). By using a single polymerase chain reaction product, genes of interest can be directionally cloned in all vectors within four different rare restriction sites at the 5′end and multiple cloning sites at the 3′end. In this way, a number of different fusion tags but also signal peptides can be incorporated at the N- and C-terminus of proteins, facilitating their expression, solubility and subsequent detection and purification. Fusion tags can be efficiently removed by treatment with site-specific peptidases, such as tobacco etch virus proteinase, thrombin, or sentrin specific peptidase 1, which leave only a few extra residues at the N-terminus of the protein. The combination of different expression systems in concert with the cloning approach in vectors that can fuse various tags makes the pCri System a valuable tool for high throughput studies.

Uptake and Metabolism of Antibiotics Roseoflavin and 8-Demethyl-8-Aminoriboflavin in Riboflavin-Auxotrophic Listeria monocytogenes

“…The gene lmo0728 was amplified by PCR using the modifying oligonucleotides 5=-ATA GCC ATG GAA GTA TCG CAT GTA AC-3= and 5=-TAT CGG ATC CTT ACT CGG AAA GTT CGT TTϪ3=. The resulting PCR product was digested and ligated to pET-MBP-1a (20), and the novel plasmid was named pET-MBP-lmo0728. A linker (5=-CAT GGT GGC GG TGG CGG TGG C-3=; 5=-CAT GGC ACC GCC ACC GCC ACC-3=) representing the tobacco etch virus (TEV) protease recognition site was introduced into the NcoI site of pET-MBP-lmo0728, resulting in pET-MBP-lmo0728TEV, which in turn was used to transform E. coli BL21(DE3).…”

Optogeneticlacoperon to control chemical and protein production inEscherichia coliwith light

“…Therefore, we reconstructed OptoLAC1 into a pACYC-derived 29 backbone, resulting in OptoLAC1B (pMAL658), which is compatible with pET and pCri and is specifically designed to control protein production optogenetically in OptoBL (Supplementary Table 1). Finally, we adapted pCri-8b, which was originally designed to assay IPTG-induced yellow fluorescent protein (YFP) production from the P T7 promoter 30 , by removing its constitutive copy of lacI 31 (see Methods), resulting in pC85 (Supplementary Table 1).…”

“…We transformed electrocompetent OptoBL cells with pMAL658 (OptoLAC1B) and pC85 (P T7 -YFP, P lacIQ _ lacI ), resulting in EMAL284. As an IPTG-inducible control containing constitutive lacI , we transformed BL21 DE3 with pCri-8b 30 , resulting in EMAL283.…”

Restriction site free cloning (RSFC) plasmid family for seamless, sequence independent cloning in Pichia pastoris

“…We applied the RSFC cloning strategy to design a plasmid family for P. pastoris allowing seamless fusions of a GOI with various tags and fusion proteins in N- and C-terminal position. There are different expression plasmids available for P. pastoris based on various cloning strategies such as Gateway [ 8 ], TA cloning [ 22 , 25 ], sticky end type IIS ligations (plasmids by BioGrammatics, ‘Electra’ plasmids by DNA2.0) and ‘classical’ typeII RE/ligation based systems ([ 30 – 32 ] and P. pastoris plasmids by Life Technologies, Carlsbad, CA, USA). The pCri vector family [ 32 ] is a multi-host platform, allowing to clone a single PCR product via restriction digestion and a MCS into different vectors.…”